Understanding the mechanisms of an effective neutralizing antibody response to HIV is one of the highest priorities in the field of HIV-specific immunity. In this regard, the inability of the humoral response of most vaccinees to cross-neutralize multiple strains of HIV is believed to be a major obstacle to the design of effective vaccines. In 2006 we observed that sera from subpopulation of our chronically infected cohorts had considerable neutralization breadth extending across clades. Much of the work done prior to that time had not focused on patients selected for broadly cross-neutralizing antibodies to HIV-1. In addition, a relatively small number of monoclonal neutralizing antibodies existed at the time. For these reasons we recruited a cohort of individuals screened for broadly cross-neutralizing antibodies to HIV. We, in collaboration with investigators at the Vaccine Research Center (VRC), used these sera to systematically dissect the means by which these patients cross-neutralize. We thus far have identified 30 such patients and are continuing to accrue additional subjects. A number of fundamental questions had not been addressed with regard to the HIV-specific humoral immune response of these patients. For example, it was not known if these patients had genetic or clinical characteristics, or HIV-specific cellular immune response characteristics in common. It was also not known whether neutralization was mediated by a few B cell clones directed to conserved epitopes or by an extremely polyclonal response to many epitopes. Given that our patients are infected with clade B viruses and should not have experienced infection by viruses belonging to multiple clades, we hypothesized that cross-neutralization is mediated through conserved epitopes on HIV envelope (Env). In addition, although considerable work had been done on patient sera, very little had been done on HIV-specific B cells. The phenotype and immunoglobulin class of HIV-specific B cells in comparison to responses to other viruses remained poorly defined. Further, it remained unclear whether patients with broad cross-neutralizing activity are unique with regard to these parameters. One primary objective of our work on the humoral response to HIV is to understand the basis of a broadly cross-neutralizing antibody response in our patients. It was not known whether there are common features of the humoral response of such patients with regard to specificity. It was also not known whether neutralization was mediated by a few B cell clones directed to conserved epitopes or by an extremely polyclonal response to many epitopes. To dissect the specificity and diversity of epitopes targeted by the B-cell response in patients with broad sera, we have initiated a collaborative effort to isolate monoclonal antibodies. To further understand the specificities and binding characteristics that underlie a broadly neutralizing antibody response we recently developed techniques that permitted isolation of human monoclonal antibodies without previous knowledge of specificity. In this technique peripheral blood memory B cells are sorted and expanded for 13 days with interleukin (IL)-2, IL-21 and CD40-ligand expressing cells. The supernatants of large numbers of micro-cultures of these cells can then be screened for neutralizing activity in a high-throughput manner. From the cultures that exhibit anti-HIV neutralizing activity the immunoglobulin genes can then be isolated, re-expressed, and characterized. We previously isolated an antibody, designated 10E8, which is among the most broad thus far described. It binds the gp41 membrane-proximal external region (MPER) of Env. It is very potent and neutralizes 98% of tested viruses. The structure of 10E8 in complex with the complete MPER revealed a site of vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues just before the transmembrane region. These data indicate that the highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies. This suggests that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 envelope glycoprotein. This antibody has also been shown to mediate potent protection against lentiviral challenge in vivo. Investigators at the VRC have used 10E8 in passive transfer experiments in Rhesus macaques. 10E8 had a similar half-life in vivo compared to the potent, broadly neutralizing antibody VRC01. 10E8 also provided protection from mucosal challenge with a simian immunodeficiency virus expressing an HIV Envelope glycoprotein. An anti-CD4 antibody did not provide protection in vivo. These results suggested that such broadly neutralizing antibodies can mediate potent protection in vivo and provide a rationale for use of these antibodies in prophylaxis or induction of these antibodies by vaccines to prevent HIV infection. More recently we have discovered new broadly neutralizing antibodies that bind novel epitopes on the HIV Env. One such antibody is 35O22 that neutralizes by binding a conserved face on contiguous areas of gp41 and gp120. This is part of a new class of antibodies that bind across the gp41-120 interface. Samples from patients within our cohort have also been used by our collaborators at the VRC to find novel sites of vulnerability on Env. For example, the antibody VRC34.01 targets a novel site on the fusion peptide and blocks HIV entry by inhibiting conformational changes in Env. Once antibodies are isolated, deep sequencing of B cell transcripts is providing important information regarding variants that may be more broad or potent, and how broadly neutralizing antibodies developed in the patient. Through this work we continue to provide a better understanding of the specificities and functions of the HIV-specific humoral immune response that are likely to provide protection from infection. Over the coming years we anticipate that this work, in the context of work from other groups, will greatly enhance our knowledge of what features of this response should be induced in vaccination strategies.